Since the discovery of RNA interference (RNAi), a crucial need has arisen for small synthetic RNAs, in particular small oligoribonucleotides with a length of 21 nucleotides (siRNA), for biological research and therapeutic applications.
The ribonucleotide units constituting these RNAs can be natural ribonucleotide units or ones that have been modified, whether by modification of the nucleic acid base or of the ribose ring, as known in the prior art, and in particular as described in Watts, J. K. et al., Drug Discovery Today, Vol. 13, 19/20, October 2008, Schram K. H. et al., Mass Spectrometry Reviews, 1998, 17, 131-251, and Porcher et al., Helvetica Chimica Acta, Vol. 88, 2005, pages 2683-2704.
Compared with the synthesis of DNA, the production of synthetic RNAs is more complex owing to the presence of the hydroxyl function in position 2′ of the ribose sugar that has to be protected. Finding the ideal protecting group is a crucial point for successful RNA synthesis. In addition to the lower coupling yields in assembly of the chain in comparison with the synthesis of DNA, the main difficulty in RNA chemistry arises from the instability of RNA in a basic medium.
That is why it is generally assumed in this field that the standard synthesis strategy used for the production of oligodeoxyribonucleotides (small DNAs), where all the reactive functions of the DNA are protected with protecting groups that are base-labile, i.e. are removed at the end of the process of chemical elongation by a single treatment with a base, is not applicable to the production of oligoribonucleotides (small RNAs).
Just as for the synthesis of DNA, the two routes most commonly used at present for synthesizing RNAs by the chemical route are, on the one hand, the route with phosphoroamidites, and on the other hand, the route with hydrogen phosphonates (H-phosphonates).
In the route with phosphoroamidites, monomers functionalized at 3′ by a phosphoroamidite group are assembled, the assembled RNA then having a 3′-5′ internucleotide phosphate linker protected, preferably, by a cyanoethyl group.
The trimethylsilylethyl (TSE) group can also be used as the protecting group for phosphates, as taught for example in Parey et al. “First Evaluation of Acyloxymethyl or Acylthiomethyl Groups as Biolabile 2′-O-Protections of RNA”, Organic Letters, 2006, Vol. 8, No. 17, 3869-3872. However, it is stated in this document that the TSE group was selected because, in contrast to the cyanoethyl group, it is not removed in basic conditions but by fluoride ions. It is also stated in this document that in reality the TSE group was removed by the iodine solution used for the oxidation carried out for obtaining the 3′-5′ phosphodiester linkers.
In the route with hydrogen phosphonates, monomers functionalized at 3′ by a hydrogen phosphonate monoester group are assembled, the assembled RNA then having a 3′-5′ internucleotide linker, which is a hydrogen phosphonate diester linker, which is then oxidized to phosphate. In this route, the RNA obtained at the end of elongation and after oxidation has unprotected 3′-5′ phosphate internucleoside linkers.
In the route with phosphoroamidites, it is generally assumed in the art that protection of the hydroxyl in position 2′ of the ribose sugar must not be effected with a base-labile protecting group that would be removed at the same time as the protecting group of the phosphate, which would lead to nucleophilic attack by the hydroxyl in position 2′ of the phosphorus atom in the internucleotide linkers resulting in 2′-5′ isomerization of the natural 3′-5′ linkers or in rupture of the 3′-5′ linker in the conditions of basic deprotection.
Thus, T. KEMPE et al., in “Nucleic Acids Research”, 1982, 10, 6695-6714, reported very low yields in synthesis of oligoribonucleotides by protecting the hydroxyl group in position 2′ of the ribose, with an acyl group such as an acetyl or benzoyl group.
The tert-butyldimethylsilyl (TBDMS) group is certainly the most used for protecting the hydroxyl at 2′ of the ribose. Although several protecting groups have been proposed for replacing it, such as the triisopropylsilyloxymethyl (TOM), bis(2-acetoxyethyloxy)methyl (ACE), tert-butyldithiomethyl (DTM), 1-(2-cyanoethoxy)ethyl (CEE), 2-cyanoethoxymethyl (CEM), 2-(4-toluylsulfonyl)ethoxymethyl (TEM), levulinyl and 2-cyanoethyl groups, most of these groups are, like TBDMS, removed by fluoride ions. However, deprotection by fluoride ions is a major obstacle for obtaining pure oligoribonucleotides because of their contamination with salts, leading to long additional procedures for purification.